FIGURE 2.
Generation of Mthfd1gt mice. A, C1THF synthase consists of three domains: the MTHFD/MTHFC catalytic domain, the MTHFD/MTHFC NADP-binding domain, and the FTHFS catalytic domain. The amino acid residues forming each domain are indicated. The Mthfd1 gene consists of 28 exons. The gene trap vector inserted into intron 26–27 of the Mthfd1 gene. PCR priming sites are indicated by arrows. B, the gene trap vector cassette in the Mthfd1 gene is detected as a 320-bp PCR product, whereas the wild-type allele is detected as a 553-bp PCR product. C, Western blot analysis of liver, kidney, brain, and colon from Mthfd1gt mice. Tissue lysates were probed with polyclonal sheep anti-mouse C1THF synthase antibody and mouse anti-human glyceraldehyde-3-phosphate dehydrogenase antibody, which served as a loading control. FTHFS, formyltetrahydrofolate synthetase; MTHFC, methenyltetrahydrofolate cyclohydrolase; MTHFD, methylenetetrahydrofolate dehydrogenase; THF, tetrahydrofolate.